Academic literature on the topic 'Bulls Spermatozoa Storage'

Effective selection and breeding in dairy farming is impossible without artificial insemination, results of artificial insemination largely depend on the quality of semen of bulls-improvers. Objective. To ascertain parameters of sperm productivity and fertilization capacity of spermatozoa of Holstein stud bulls of foreign selection in conditions of LLC “Ukrainian Genetic Company”. Methods. We have analyzed ejaculates of 20 mature Holstein stud bulls elite-record class brought to animal breeding enterprise from Germany and the Netherlands. The duration of use of bulls at the breeding enterprise is on average 4–5 years.Quantitative indicators of sperm were taken per year to avoid the influence of seasonal factors. Semen was collected twice a week by artificial vagina on the false stud. Ejaculates obtained were analyzed according to the DSTU 35.35-97 incertified production laboratory of LLC “Ukrainian Genetic Company”, equipped with up-to-date equipment for sperm quality assessment, packing, freezing and storage. Quantity and quality characteristics of sperm were analyzed on computer sperm analyzer IVOS (HamiltonThorneResearch, USA). Sperm fit for cryopreservation is diluted at a temperature of +35°С in the environment AndroMed (Germany) at rate no less than 20 million spermatozoids for one sperm dose and packed into 0.25 ml straws. Frozen sperm doses after check are kept in the special bio-storage in liquid nitrogen at a temperature of -196°С. Results. Fertilization capacity ofthe spermatozoaof experimental stud bulls was measured by the percentage of quantity of cows and female calves that were impregnated after the first insemination in Zhytomyr (PAF “Ierchyky”, SPDG “Nova Peremoga”, ALLC “Ptakhoplemzavod “Korobivskyi”) and Kyiv (LLC “Agrofirm “Kyivska”) regions. On the whole results of insemination of 12525 cows and 1230 female calves of puberty age were analyzed. Sperm productivity index and average fertilization capacity of spermatozoa of stud bulls was assessed by the method of M. M. Maiboroda, S. H. Hermanchuk, Yu. P. Polupan, and D. M. Basovsʼkyy. It was revealedsignificant variability of sperm productivity indicators of experimental stud bulls, which vary within: number of obtained high-quality ejaculates within one year –32–173 pcs., total volume of native sperm – 201–1016 ml, average value of spermatozoa concentration in 1 ml – 1.51–3.52 milliard, average sperm motility in ejaculate – 7.2–83 points. Bull studs of LLC “Ukrainian Genetic Company” are characterized by a rather high index of sperm productivity which equals from 5.19 to 15.29 billion of motile spermatozoids in ejaculate. Average fertilization capacity of spermatozoa of experimental Holstein bulls in conditions of 4 farms of Zhytomyr and Kyiv region ranges from 37.1 to 61.4%, average figure for examined livestock is 50.9%. During the research we found that stud bulls whose sperm fertilization capacity was above 50% in most cases (6 out of 8) had index of sperm productivity above average – 9.86–15.25billion of motile spermatozoids in ejaculate. We have detected positive and statistically significant correlation between fertilization capacity of spermatozoa and the average value of concentration, motility of spermatozoids in ejaculate (+0.474 and+0.417 respectively) and sperm productivity index (+0.639). Conclusion. Our research proves dependence of fertilization capacity of sperm on its quality parameters. The results we obtained suggest that it is expedient to use index of sperm productivity to assess quality of sperm and prove its importance for breedingbulls selection.

SummaryThe aim of this work was to examine the influence of cryostorage duration of Pinzgau bull's insemination doses (IDs) on some sperm traits. The IDs were frozen by a slow freezing method and stored in liquid nitrogen for different periods: less than 8 years (group 1), 8–13 years (group 2) and 14–18 years (group 3). Motility (CASA), pathological sperm rate (Giemsa staining), apoptotic (Yo-Pro-1-positive) and necrotic (propidium iodide-positive) cell occurrence and fertilizing ability (penetration/fertilization test) of spermatozoa were evaluated post-thaw. The average post-thaw sperm motility in all examined groups was over 40%. No significant influence of storage length either on the sperm total motility or progressive movement was revealed. In each tested group the average rate of malformed spermatozoa did not exceed 20%. No effect of cryostorage length on the occurrence of apoptotic or necrotic sperm was noted. Similarly, penetrating/fertilizing ability of sperm did not differ among the groups, excepting differences in the rate of pronuclei (PN) formation. In group 1, 72.9% of eggs showed two visible PN following 20 h incubation with sperm, whilst in groups 2 and 3 only 67 and 54.5% of zygotes, respectively, had both PN at this time. These results revealed no influence of storage time on the bull spermatozoa in all parameters excepting the rate of PN formation. As high inter-male variability was observed in the susceptibility of bull sperm to cryostorage, individual differences should be taken into account when semen from individual bulls is to be stored for a long time.

The aim of the work was to examine the effect of modified TALP medium (TALP/Pp, Animal Pharma B.V., Hengelo, The Netherlands)—used in the sperm sexing procedure—on bull sperm membrane status. The TALP was modified by replacement of bovine serum albumin (BSA) with a mixture of several plant proteins and soya lecithin (Pp). The Pp component was prepared using a high pressure homogenization process. The TALP/Pp had the same pH and osmotic pressure as the original TALP medium (TALP/BSA). The work was divided into 2 parts: (1) Nine ejaculates collected from 2 bulls (Holstein and Polish Red) were used. Immediately after collection, each ejaculate was split into 2 parts and diluted (1:2) with TALP/BSA or TALP/Pp. The sperm membrane status was examined after 3 days of storage at 15�C. (2) Fifteen ejaculates collected from 5 bulls (Holstein, Polish Red, and Simmental) were used. Each ejaculate was split into 2 parts: the first part was diluted with TALP/BSA, stained, incubated, and sexed according to the XY Inc. bull semen sexing procedure; the second part was diluted, stained, incubated, and collected after sexing into TALP/Pp with no egg yolk addition. In both groups no red food due was used to identify and exclude the dead spermatozoa from the sorted fractions. The sperm sexing procedure was performed with an SX MoFlo high-speed sorter at a speed of 3000–4000 cells/s. After collecting about 10 million spermatozoa, both fractions, X andY, were mixed, centrifuged at 700g for 15 min to concentrate the spermatozoa (20 million mL–1), and the sperm membranes examined. For sperm membrane examination, 'live/dead' samples were stained with SYBR-14/propidium iodide fluorochromes and analyzed by flow cytometry. The data from 20 000 spermatozoa were collected for each sample. The percentage of membrane-intact ('live') spermatozoa was taken for statistical analysis. The mean percentage of live spermatozoa stored for 3 days in TALP/BSA v. TALP/Pp was 25.7% (SD = 7.48) v. 28.58% (SD = 7.04), respectively (P < 0.01). The mean percentage of live spermatozoa in samples of sexed semen was 33.57% (SD = 18.97) for TALP/BSA and 38.51% (SD = 20.22) for TALP/Pp (P < 0.01). It can be concluded that Pp should be considered as a replacement for BSA in the TALP medium used for bull sperm sexing because (1) it results in significantly higher numbers of live spermatozoa after storage and/or sexing; (2) it eliminates a possible source of transmissible diseases (such as bovine spongiform encephalopathy); and (3) it decreases the total cost of the basic media used for the bull sperm sexing procedure.

This research was conducted at the Teaching Farm of the Faculty of Veterinary Medicine, Airlangga University, to know the effect of age, temperature, and humidity on the quality of semen of bulls which are turned into frozen semen. This research uses fresh semen samples of bulls of various ages grouped into 3 groups, young, middle age, and old groups and measures temperature and humidity during the storage process. Fresh semen is collected using an artificial vagina and then examined macroscopic and microscopic tests which is then checked for post thawing motility. Data analysis uses Analysis of Variants (ANOVA) followed by Duncan. The results showed that the percentage of spermatozoa motility of fresh semen and post thawing differed and age affected the quality of semen produced. There was no effect of temperature and humidity when processing of collecting semen. Bull’s semen at Teaching Farm Faculty of Veterinary Medicine Airlangga University has good quality and is suitable for Artificial Insemination.

The competitiveness of buffalo breeding will depend on the utilization of reproductive biotechnologies that allows acceleration of genetic progress. A major factor hampering the efficiency of both artificial insemination and in vitro embryo production programs in this species is male hypofertility. Reports for several species suggest that seminal plasma contains factors that influence male fertility. Osteopontin is a glycoprotein found in several biological fluids including seminal plasma, and its presence is associated with spermatozoa concentration. In cattle, expression of osteopontin was highly correlated with bull fertility, and it was proposed to be a marker to predict male fertility (Cancel et al. 1999 Biol. Reprod. 60, 454–460). No data are available about the presence or activity of osteopontin in water buffalo. The aim of this preliminary study was to determine if osteopontin is present in buffalo semen and to evaluate whether freezing procedures cause the loss of osteopontin from spermatozoa. Semen was collected in authorized semen collection centers from 6 buffalo bulls by using an artificial vagina. A collection of bovine semen was used as a positive control. An aliquot from each sample was frozen using standard procedures for semen storage. Each ejaculate was centrifuged at 600g for 10 min at room temperature, and the supernatant was recovered and centrifuged at 10 000g for 1 h at 4�C. The total protein concentration in seminal plasma and spermatozoa was determined by the Bradford method, using ovoalbumin as the standard. Proteins (50 �g) were separated by electrophoresis and analyzed by western blotting (Cancel et al. 1999). Polyclonal antibodies against bovine milk osteopontin were prepared as previously described (Cancel et al. 1997 Biol. Reprod. 57, 1293–1301). The intensities of bands indicated by western blot were quantified by densitometer. Osteopontin was detected in all samples of buffalo semen. Most of the osteopontin detected was in the seminal plasma. Relative amounts of osteopontin detected in spermatozoa were 50% or less of that detected in seminal plasma; furthermore, the protein was not found in sperm from all bulls. These results suggest that most osteopontin is produced by the ampullae and seminal vesicles, similar to what was reported for cattle (Cancel et al. 1999). Semen frozen by standard procedures showed a reduction in amount of osteopontin by up to 50%. These studies suggest that the fertility-associated protein osteopontin may be useful as a sensitive tool to evaluate whether sperm storage procedures are detrimental and result in excess loss of osteopontin from sperm. In conclusion, the results have demonstrated that osteopontin is present in buffalo seminal plasma and sperm. Further studies will examine whether the expression of osteopontin is correlated with the fertility of buffalo bulls, as has been demonstrated in bovine bulls.

Despite being subject to prior assortment, frozen bull sperms commercialized for artificial insemination may present certain morphological defects. The present study aims (i) to assess the artificial insemination success of the most common cattle breeds in Algeria and (ii) to evaluate the possible effects of commercialized bull’s semen quality on this operation. Artificial insemination was assessed through four years of field monitoring by inseminating different cattle breeds of normal fertility. However, semen quality was evaluated using light microscopy by measuring viability, motility, and morphological abnormalities of spermatozoa. Field study revealed a high percentage of normal calving in red and white Holstein breed (44.83 %) against the high percentage of embryonic mortality (46.43 %) and calving with a malformation (10.71 %) in Montbéliarde breed. Semen quality assessment revealed that sperm viability and motility were higher in Holstein breeds than in Montbéliarde. Furthermore, significant differences between semen bulls were found in the proportion of abnormal spermatozoa; a higher rate of sperms with the abnormal head was observed in the black and white Holstein breed (69.3±10.98 %). However, the percentage of abnormal sperms with tail defects was higher in the Montbéliarde breed (67.5±10.74 %). The lousy quality of the selected semen and/or the poor handling and storage of frozen semen constitute a determinant factor that hinders the success of artificial insemination in the arid region of Tiaret (Algeria).

Background: Pasundan cattle are local cattle native to Indonesia. One way to conserve beef cattle genetics is to use Artificial Insemination technology. The success of Artificial Insemination can be influenced by the quality of semen. Purpose: To determine factors affecting fresh semen quality in Pasundan cattle at UPTD BPPIBTSP Ciamis. Methods: The data were obtained through observations on seven Pasundan bulls in March 2021 towards fresh semen quality and some factors influencing it. The Pasundan bulls observed were seven productive males. Results: The fresh semen quality of Pasundan cattle, such as volume, color, and pH, showed good result,s but the average consistency and concentration of spermatozoa were still below the standard. The factors that can affect the fresh semen quality are the breed of beef cattle, age, body weight, feed, season, exercise, and frequency of semen storage. Conclusion: The determining factors that can cause the consistency and concentration of Pasundan cattle’s spermatozoa at UPTD BPPIBTSP Ciamis are feed and season.

In order to preserve male germoplasm, the recovery and cryopreservation of spermatozoa from the epididymides of hunted animals represents an accessible source of gametes. As a first experimental model, epididymal spermatozoa from slaughtered bulls were recovered at 30, 54, 78 and 102 h after death. The scrotal contents were stored at either 5 or 20°C. The sperm cells of each treatment (time + temperature combinations) were frozen with Triladyl (T) or Triladyl + Trehalose (TT) diluents. In order to assess sperm viability and integrity, post-thawing evaluation included individual motility, supravital stain, hyperosmotic swelling test (E+), acrosome status and sperm chromatin structure assay. Both at raw and post-refrigerated states, the sperm motility rate was higher in sperm obtained from epidydmes stored at 5°C, compared with those stored at 20°C for all collection times. Sperm collected at 102 h after death from epididymides stored at 5°C maintained a motility of 20% (120 h, raw state). When comparisons were carried out after thawing, motility was higher in the 5°C group, achieving the best results with TT diluent (7.5%) at 102 h. However, when supravital stain and E+ tests were observed, viability and membrane integrity were well preserved even at 102 h post mortem (30 and 36%, respectively, with TT diluent at 5°C). These results suggest that frozen-thawed epididymal spermatozoa could have a low motility rate while most of them remain alive. Acrosome status was not greatly affected by storage time. In a second experiment, epididymal spermatozoa from hunted red deer stags (Cervus elaphus) were recovered at 4 and 30 h after death. The scrotal contents were stored at 20°C, because that temperature is closer to field and shipment conditions. The sperm cells were frozen with TT diluent. Post-thawing evaluation included the same parameters indicated for bull spermatozoa. The assessment of spermatozoa collected at 30 hours post mortem and then subsequently frozen and thawed indicated that at this time an acceptable motility rate (35%) and viability (39.7%) were achieved. Frozen and subsequently thawed epididymal spermatozoa showed 47.9% of membrane integrity, 59.3% of acrosome integrity and 26.5% of chromatin damage, using TT diluent. A preliminary in vivo trial demonstrated that the pregnancy rate in artificially inseminated deer decreased when sperm were obtained at 30 h post mortem. According to these results, it may be concluded that storage at 5°C is better than 20°C to obtain well preserved epididymal spermatozoa from bulls, and that TT could be a useful cryoprotectant to preserve viable and fertile sperm cells after the freezing–thawing process. Before these results can be applied to assisted reproduction programs in endangered deer species, some adaptations must be developed.

Altering the lipid composition of sperm plasma membranes affects sperm cryosurvival. Cryopreservation induces many stresses on the spermatozoa, including destabilization of the plasma membrane, which results in the loss of sperm motility and function. Treating bull spermatozoa with cholesterolloaded cyclodextrin (CLC) prior to cryopreservation increases sperm cryosurvival rates. This study compared the effect of adding other sterols, which should incorporate into the membrane and increase membrane fluidity at low temperatures, thereby increasing cryosurvival. Ejaculates from four bulls were divided into two experiments (E). In E1, ejaculates were extended with Tris, and then subdivided into four treatments: No additive (control), 1.5 mg CLC/120 million sperm (positive control), and 1.5 mg/120 million sperm in cyclodextrin pre-loaded with either cholestanol or desmosterol. Spermatozoa were incubated for 15 min at 22�C after which both the ability of fresh spermatozoa to bind to the zona pellucida (ZP) and chicken egg perivitelline membrane (EPM) and their osmotic tolerance were evaluated. In E2, sperm were diluted to 120 million cells mL–1 in a Tris diluent and treated as described for E1. Then, samples were diluted 1:1 (v:v) in Tris with 20% Egg Yolk (EY) and cooled to 5�C. After dilution 1:1 (v:v) with Tris containing 10% EY and 16% glycerol, samples were allowed to equilibrate for 15 min, and then were packaged into 0.5-mL straws, frozen in static liquid nitrogen vapor for 20 min, and plunged into liquid nitrogen for storage. Straws were thawed and the motility and zona-binding ability were determined using a Hamilton Thorne Motility Analyzer (Hamilton Thorne Biosciences, Beverly, MA, USA) and epifluorescence microscopy, respectively. Treatment differences for sperm motility, osmotic tolerance, and zona binding were determined using analysis of variance. Treating spermatozoa with CLC resulted in more fresh bull spermatozoa binding to the EPM and ZP compared to cholestanolor desmosterol-loaded cyclodextrin-treated spermatozoa or control cells (P < 0.05). No differences were observed between EPM and ZP binding (P > 0.05). The percentages of total and progressively motile spermatozoa were higher for fresh samples treated with cholesterol-, cholestanol-, or desmosterol-loaded cyclodextrin than for control cells (P < 0.05) when spermatozoa were exposed to anismotic conditions, and then returned to isosmolality. After cryopreservation, the percentages of motile spermatozoa and number of spermatozoa binding to ZP were similar for spermatozoa treated with CLC (56% and 115 sperm/ZP) and cholestanol (53% and 108 sperm/ZP) compared to spermatozoa treated with desmosterol (42% and 86 sperm/ZP; P < 0.05). All treatments provided higher motility and binding efficiency than control spermatozoa (32% and 62 sperm/ZP; P < 0.05). Therefore, adding cholesterol or cholestanol to bull sperm membranes improved cell cryosurvival. Studies to determine if cholestanol affects sperm capacitation need to be conducted.

Assisted reproductive techniques (ART) are widely used in domestic species, with increasing applications in wildlife conservation. However, the currently available techniques for semen preparations are not fully reliable or applicable in all species to ensure successful ART outcomes. Recent developments in nanotechnology offer new horizons for further optimization of sperm preparations. The use of conjugated magnetic nanoparticles allows for selective targeting and removal of moribund spermatozoa through the technique known as nanopurification, which has shown to be beneficial in domestic boars and bulls. Nanopurification is a rapid, straightforward, effortless, and noninvasive technique that, when applied in wildlife or endangered species, is expected to have a tremendous impact on ART outcomes. In this study, we evaluated the effectiveness of magnetic (iron oxide) nanoparticles designed to target moribund spermatozoa (e.g. acrosome reacted) on domestic felid and threatened bovid species. Fresh epididymal spermatozoa of domestic cats and ejaculated spermatozoa from genetically valuable threatened bulls (Ankole and Sahiwal) were collected and mixed with iron-oxide magnetic nanoparticles (MNP). After 30min of incubation at 38°C, semen mixtures were placed on an electromagnetic field (10-15min) to trap MNP-bound spermatozoa (moribund), followed by the elution of viable nontrapped spermatozoa (nanopurified). All samples were analysed for motility characteristics using a computer-assisted sperm analyzer. Aliquots of cat spermatozoa were subjected to (1) morphology and acrosome analyses (n=4-8 replicates), (2) cryotolerance to liquid nitrogen storage (n=4 replicates), and (3) IVF (n=1 replicate). Data were analysed with Student’s t-test and P&lt;0.05 indicated significant differences. Regardless of the species, both control and nanopurified spermatozoa revealed comparable motility characteristics (P&gt;0.05). Sperm MNP-bound samples exhibited higher proportions of moribund spermatozoa as compared to their nanopurified and control nonpurified counterparts (57.5v. 47.5 and 49.5%, respectively; P&lt;0.05). Cryopreservation revealed a trend for higher post-thaw motility of nanopurified cat spermatozoa (18%) v. the controls (12%). The use of frozen-thawed control v. nanopurified cat spermatozoa to in vitro fertilize oocytes (n=13 per group) resulted in comparable embryo cleavage (15v. 31%, respectively) and blastocyst (15v. 8%, respectively) rates. This preliminary study indicates the successful interactions of MNP nanoparticles with feline and bovine spermatozoa. The nanopurification seems to improve the cryotolerance of cat spermatozoa without affecting their fertilization potential. Additional research is being conducted to confirm the current findings and optimize this novel technique for future implementation in conservation breeding programs. Work supported by USDA-ARS 58-6402-3-018 and Morris Animal Veterinary Student Scholarship - D18ZO-608.

MSCAGR (Animal Science)
Department of Animal Science
In order to realize many of the potential advantages of AI, storage of semen is necessary. Semen storage is only possible using a system that decreases and/or halts the metabolic processes of the spermatozoa, allowing no significant loss of fertility. Numerous factors affect the success of spermatozoa storage. This study was designed to compare the effects of egg yolk, soybean milk and coconut water in Tris extender using different storage methods for Nguni bull spermatozoa storage. Bull semen was collected from two adult Nguni bulls approximately four years old and kept under similar managerial conditions. Using electro-ejaculator, semen was collected from each bull into a graduated semen collection tube. Macroscopically evaluation of the sample was performed immediately after collection. Only the semen free from contamination was processed. The kinetic properties namely: total spermatozoa motility, and progressive spermatozoa motility were analysed using CASA. Semen sample was stained and spermatozoa morphology and vitality also analysed using CASA. The extended semen was then split into three groups. The first group was stored at room temperature (25 °C). The second group was cooled to 4 °C and stored in the refrigerator. The third group was also cooled to 4 °C for 2 h in the refrigerator, then held in LN2 vapour 5 cm above the surface of LN2 at ~ -80 °C for 10 min and then plunged into LN2 for storage at -196 °C. Different colours of straws and plugging powder were used for identifying each extender. After 3 days of storage at room temperature, in the refrigerator and in LN2, the extended semen was split into three portions and assayed for kinetic properties using the first portion. The second portion was assayed for spermatozoa morphology and the third portion for spermatozoa vitality. The results from the fresh semen extended with all three extenders (TEYE, SBME and COWE), and analysed immediately after dilution at room temperature (25 ºC), showed no significant difference (P > 0.05) in the mean values of the kinetic and morphologic properties and viability, on spermatozoa TM, PM, AR, AT, CT; BT and LS. After three days of storage, there was no significant difference (P > 0.05) in the kinetic morphologic properties and viability of semen stored at room and refrigeration temperature regardless of the extender in use. There were, however, significant differences (P < 0.05) in the TM, PM, AR and DL of the frozen semen samples. For the short storage period of semen used for AI, from this study, it is recommended that semen should be kept at room or refrigeration temperature regardless of the three extenders used. However, for long storage of frozen semen TEYE is recommended. The egg yolk-based extender provided greater preservation of motility and bull spermatozoa integrity during the freezing process than did SBME and COWE.